1. Field of the Invention
The present invention relates to a chemically modified paper adapted to immobilize nucleic acid residues and proteins and a method for producing and utilizing the same.
2. Description of the Prior Art
Living cells contain nucleoproteins which are proteins combined wth natural polymers, called nucleic acids. Nucleic acids have a long chain backbone attached to which are various groups, which by their nature and sequence, characterize each nucleic acid. The backbone of protein is a polyamide chain, whereas the backbone of the nucleic acid molecule is a polyester chain; a polynucleotide chain. The ester is derived from phosphoric acid and sugar.
The sugar is D-ribose for the nucleic acids known as ribonucleic acids (RNA) and the sugar is D-2-deoxyribose in the group known as deoxyribonucleic acids (DNA). Attached to each sugar, through a beta-linkage is one of a number of heterocyclic bases. A base-sugar unit is a nucleoside; a base-sugar-phosphoric unit is a nucleotide.
The bases in DNA are adenine and guanine, which contain the purine ring system; and cytosine, thymine and 5-methylcytosine, which contain the pyrimidine ring system. RNA contains adenine, guanine, cytosine and uracil. The proportions of these bases and their sequence along the polynucleotide chain differ from one kind of nucleic acid to another.
DNA consists of two polynucleotide chains wound about each other to form a double helix. The helixes are bound to each other at intervals by hydrogen bonding between the bases. The nucleic acids, as DNA, control heredity on the molecular level. The genetic code is stored as the sequence of bases along the polynucleotide chain, consisting of permutations of adenine (A), guanine (G), thymine (T) and cystosine (C). For RNA, uracil (U) is substituted for thymine (T).
In most entities genetic information is channeled from DNA into messenger RNA (mRNA) and then translated with the help of transfer RNA (tRNA) into polypeptides. The transfer of information from DNA to mRNA is known as transcription and occurs when a mRNA strand complements a part of one DNA strand. Transfer RNA brings specific amino acids to a ribosome - associated, mRNA template, holds them there while the amino acids are joined to a polypeptide structure and is then released by enzymatic acid from the polypeptide chain. RNA is also found in ribosome particles which consist of ribosomal RNA (rRNA) and protein. Both tRNA and rRNA have double-stranded regions as a result of folding back onto itself.
As employed herein the term "residue" includes an entire strand or chain and parts of said strand or chain of nucleic acids, such as RNA or DNA. The complementary binding of mRNA to a strand of a cell's DNA was demonstrated by "hybridization" tests in 1961. In conventional hybridization a residue of a nucleic acid, as mRNA residue, is separated and pulse labeled with a radioactive isotope, such as Phosphorous 32, (P.sup.32). The labeled residue is incubated with a heat-denatured (single stranded), nucleic acid, as DNA. When the DNA, for example, is from the same species as the RNA, the mRNA will hybridize with the complementary DNA strand on cooling. The non-hybridized RNA can then be destroyed by RNAase treatment, a conventional treatment, and the RNA-DNA hybrid, identifiable by the presence of P.sup.32 labeled RNA, and other tests is recovered. Hybrids are formed only when homologous DNA and RNA (having complementary bases) are employed. Similarly, labeled tRNA and rRNA from a given species hybridizes only with their homologous DNA.
In conventional hybridization testing, DNA only was immobilized on a solid matrix. For example, DNA was coupled to acetylated, phosphorylated cellulose in a chromatography column. The phosphate groups on the cellulose acetate combined with the glycosylic hydroxyls of the (sugar) deoxyribose of DNA. Another method employed agar or gel to trap denaturated DNA. Others proposed to hydrogen bond DNA residues to nitrocellulose membranes. Still others attempted to link DNA to cellulose using water-soluble carbodiimide or to link DNA to agarose, activated with cyanogen bromide.
Such prior immobilization of DNA residues has not proved entirely satisfactory. In all prior attempts to hybridize, DNA was immobilized on a matrix. RNA has not been successfully immobilized, so that a labeled DNA residue could act as a probe. This is a serious defect. Further, the relatively large amount of necessary support matrix prohibited sensitive analytical hybridization studies.
In addition, the proposed nitrocellulose matrix is a relatively unstable substrate for DNA, since only a hydrogen bond, not a covalent bond, is formed. Further, short chains or residues of DNA, when applied to nitrocellulose, are too unstable for successful hybridization. Therefore, one could not use desirable, highly fractionated DNA chains for hybridization, only relatively long chains of DNA.
Therefore, there exists a long felt need to provide a compact matrix for covalently immobilizing both long and short residues of both DNA and RNA, as well as proteins, to allow small fractions of nucleic acids or proteins to be sensitively analyzed with labeled probes. In Volume 16, J. Applied Chem. pp. 351-355 (1943) cotton fabric, not paper, was impregnated with pyridine salts of chloromethyl ethers. Upon suitable reduction and activation, the cotton fabric was dyed by coupling 2-naphthol with the diazotized fabric. Cotton fabric is not a suitable substrate for hybridization, as a column containing such fabric would have to be employed. Further, there is no teaching in the article of immobilizing nucleic acids or proteins.
For the purpose of this application reverse hybridization includes immobilizing RNA on a thin matrix and probing with labeled DNA.